Introduction

Non-steroidal anti-inflammatory drugs (NSAIDs) are widely used for the treatment of arthritis, inflammation, and pain [1]. The present drugs, like aspirin, ibuprofen, rofecoxib, naproxen, indomethacin, celecoxib, and diclofenac are some best-selling examples of NSAIDs for the treatment of painful conditions [2]. However, long-term clinical use of NSAIDs is associated with side effects such as gastric ulceration and hemorrhage [3–7]. Cyclooxygenase (COX) enzyme produces prostaglandins that promote inflammation, pain, and fever. The two closely related isoforms of cyclooxygenase are designated as cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2). COX-1 is a ubiquitous iso-enzyme that regulates physiological functions and COX-2, which is inducible primarily present at the site of inflammation [8–10]. Most of the presently available NSAIDs on the market inhibit both isoforms of cyclooxygenase [11–16]. Therefore, there is a need to develop safer COX-2 inhibitors with high selectivity profiles over the currently approved drugs, which are associated with side effects, and this is a challenging task in medicinal chemistry research.

Pyrimidine derivatives are well-known privileged nitrogen-containing heterocycles that are ubiquitous in the field of medicinal chemistry and also present in various natural products [17]. They were found to be potent agents for anti-fungal [18], anti-bacterial [19], anti-inflammatory [20, 21], anti-cancer [22–24], anti-HIV [25, 26], and anti-tuberculosis activities [27, 28]. The synthesized 4-hydroxy-2-methyl-6-(phenyl)pyrimidine-5-carbonitrile derivatives are also found to resemble many natural products (Fig. 1a–c) and have valuable biological properties [29].

Fig. 1
figure 1

Examples of some natural bioactive pyrimidines

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Due to the valuable biological significance of pyrimidine compounds, the synthesis of pyrimidine derivatives has received considerable attention. Until today, a number of methods have been reported for the synthesis of pyrimidine rings by the use of 1,3-binucleophilic centers such as guanidine, amidines, urea, and thiourea [30–34]. Recently, Rong [35] reported the synthesis of similar types of thiosubstituted pyrimidine derivatives, while Sheibani et al. [36] synthesized some pyrimidine derivatives via multicomponent reaction using MgO as a catalyst. The reported methods have their own merits and demerits.

In today’s synthetic organic chemistry, multicomponent reactions (MCRs) are used effectively and efficiently in sustainable and diversity-oriented synthesis of heterocycles [37]. These reactions play a crucial role in synthetic organic chemistry, having the ability to form carbon–carbon and carbon–heteroatom bonds [38]. MCRs have also been examined as a fast and convenient solution for the synthesis of diverse classes of compounds and are an area of considerable current interest [39–41]. During last few decades, MCRs have been used for the synthesis of a large number of bioactive heterocycles and have significant advantages over multistep synthesis for the construction of complex molecules in one-pot operation with high yield, high atom economy, shorter reaction time, and mild reaction conditions [42–44].

Having in mind the pharmaceutical importance of pyrimidine derivatives and in continuation with our research interest [45], we sought to design, synthesize, and evaluate new potential anti-inflammatory multifunctionalized pyrimidine derivatives through MCRs. Herein, we report for the first time a novel synthesis of multifunctionalized pyrimidine derivatives by using commercially available aromatic aldehydes, ethyl cyanoacetate, and acetamidine hydrochloride in DMF at 80 °C, as shown in Scheme 1.

Scheme 1
scheme 1

General reaction for the synthesis of multifunctionalized pyrimidine derivatives

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Results and discussion

Our experimental investigation focused on the synthesis and evaluation of anti-inflammatory activity of novel multifunctionalized pyrimidine derivatives. In the present protocol, we initiated our studies by employing a trial reaction of 3-nitrobenzaldehyde (1 mmol), ethyl cyanoacetate (1 mmol) using quantitative amount of sodium hydroxide in 5 ml ethanol was stirred in a 30-ml round-bottom flask at room temperature for 10 min followed by addition of acetamidine hydrochloride (2 mmol) and kept for heating at 80 °C up to completion monitored by TLC. After completion of the reaction indicated by TLC, the reaction mixture was poured in ice-cold water and neutralized by a few drops of 50 % concentration of conc. HCl by volume to volume. Unfortunately, in the trial reaction, instead of formation of the desired product, we got the hydrolyzed product of a Knoevenagel reaction. To overcome this problem, a variety of solvents with different catalysts were examined. The main problem raised in the synthesis of the desired multifunctional pyrimidine derivatives is the hydrolysis of the Knoevenagel product. It has been observed that this hydrolysis is very significant in polar medium. So using anhydrous DMF and a desired amount of NaOH, the reaction completed smoothly giving 4-hydroxy-2-methyl-6-(3-nitrophenyl)pyrimidine-5-carbonitrile in excellent yield. The use of ethanol, water, ethanol–water mixture, DMF–water mixture, and even using weak organic bases like DBU, DBN, piperidine, triethyl amine were not helpful for the said reaction. While in the case of DMSO, THF, and acetonitrile, low yield was observed.

The optimization of catalyst quantity and temperature are of crucial importance for any successful synthesis. The model reaction was further studied to optimize the temperature and quantity of NaOH. It has been noted that the faithful results were obtained in DMF at 80 °C using 0.08 g NaOH. The decrease in temperature had a significant effect on yield. The excellent yield of product was observed in the presence of an optimized amount (0.08 g) of NaOH as compared to other bases such as K2CO3 and NaHCO3. All of these experimental observations are summarized in Table 1.

Table 1 Optimization of reaction conditions
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Reaction conditions 3-nitro benzaldehyde (1 mmol), ethyl cyanoacetate (1 mmol), acetamidine hydrochloride (2 mmol) and solvent (3 ml).

The structure of the isolated product under the optimized reaction condition was confirmed on the basis of IR, 1H NMR, 13C NMR, mass spectrometry, and elemental analyses. In1H NMR and 13C NMR of desired 4-hydroxy-2-methyl-6-(3-nitrophenyl)pyrimidine-5-carbonitrile revealed the presence of incorporated methyl group of acetamidine moiety at δ 2.47 and δ 21.99 ppm, respectively. The broad signal appearing at δ 13.60 ppm in 1H NMR indicated the presence of the –OH group. The peaks observed at δ 7.86–8.67 ppm were attributed to the four protons of aromatic moiety. The presence of a sharp absorption peak in FT-IR at 2231 cm−1 indicates the presence of the –CN group in the desired compound. Finally, the mass spectrum shown base peak at m/z = 256 which is in accordance with proposed structure.

This initial success encouraged us to check the ability of the protocol by using structurally diverse aldehydes bearing an electron-donating or an electron withdrawing group showed excellent results. It has been observed that all such variations did not affect the yield and reaction time of the proposed methodology. All these results are shown in Table 2.

Table 2 Synthesis of multifunctionalized pyrimidine derivatives
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Reaction conditions Aldehydes (1 mmol), ethyl cyanoacetate (1 mmol), acetamidine hydrochloride (2 mmol), and solvent (3 ml).

Based on the above experimental results, a plausible mechanism for the reaction is predicted and shown in Scheme 2. The reaction begins with the initial formation of Knoevenagel product of aldehyde and ethyl cyanoacetate followed by Michael addition of acetamidine. The formation of desired product takes place by further intramolecular nucleophilic cyclization and subsequent aromatization to afford 4-hydroxy-2-methyl-6-(3-nitrophenyl)pyrimidine-5-carbonitrile. The used alkaline medium not only helps for promoting the reaction but also in neutralizing the used acetamidine hydrochloride. All the synthesized compounds have been confirmed on the basis of their spectroscopic data. The data is in harmony with the proposed structures.

Scheme 2
scheme 2

Plausible mechanism for the formation of 4-hydroxy-2-methyl-6-(3-nitrophenyl)pyrimidine-5-carbonitrile

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Anti-inflammatory activity

In the course of our research program, we devoted to display of new lead structures for bioactivity [46], we were interested in anti-inflammatory evaluation of multifunctional pyrimidine moieties devoid of the undesirable side effects associated with classical NSAIDs. To rationalize the applications of the synthesized compounds, the anti-inflammatory activity of all newly synthesized pyrimidine derivatives have been determined at 25 and 50 mg/kg, p.o. by carrageenan-induced rat paw edema method in Wistar albino rat and compared with diclofenac sodium as a standard reference drug. The effects of all synthesized compounds and standard reference drugs on paw edema induced by carrageenan are depicted in Table 3 and also by bar diagram (Fig. 2). The compound AC1, 2, 5, 9, and AC10 showed promising anti-inflammatory activity while the remaining compounds AC3, 4, 6, 7, and AC8 showed good activity. From the results of anti-inflammatory activity of synthesized pyrimidine derivatives, it has been found that compound AC10 shows highest anti-inflammatory activity due to presence of methyl group at fourth position of benzene ring of aldehyde moiety. The presence of the methoxy group at third position exhibit lowest anti-inflammatory activity among the synthesized compounds (AC7). The presence of a nitro group in the compound (AC1 and AC2) was also found to increase the anti-inflammatory activity.

Table 3 Effect of compound AC1 to AC10 on paw edema induced by carrageenan in rats
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Fig. 2
figure 2

Effect of ELE on paw edema induced by carrageenan in rats

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The animal studies were approved by the Institutional Animal Ethics Committee (IAEC) of SCAN research Laboratory, Bhopal, constituted for the purpose of control and supervision of experimental animals by Ministry of Environment and Forests, Government of India, New Delhi, India.

Conclusions

In the present study, the novel pyrimidine privileged scaffolds were prepared by a simple one-step MCR of structurally diverse aldehydes with ethyl cyanoacetate and acetamidine hydrochloride. Most of the synthesized compounds showed promising anti-inflammatory activity compared to diclofenac sodium. The study of these experimental results of pyrimidine moiety could be optimized in future for the development of selective COX-2 inhibitor as potential anti-inflammatory agents.

Chemistry

Experimental section

Melting points were recorded on a digital melting point apparatus and are uncorrected. IR spectra were measured using ATR on PerkinElmer spectrometer. 1H NMR and 13C NMR spectra were obtained on BRUKER spectrometer at 300 and 75 MHz, respectively, using TMS as an internal standard in DMSO solvent. Mass spectra were recorded on Shimadzu Toshvin. The chemicals were purchased from Aldrich and Alfa Aesar chemical companies. All reactions were monitored on TLC aluminum sheets Silica Gel 60 F254 under UV cabinet.

General procedure for the synthesis of AC1-10 compounds

A mixture of aromatic aldehydes (1 mmol), ethyl cyanoacetate (1 mmol), and NaOH (2 mmol) in 3 ml DMF was stirred for 10 min at room temperature. After 10 min, acetamidine hydrochloride (2 mmol) was added and kept for heating at 80 °C in an oil bath. After completion of the reaction monitored by TLC, the reaction mixture was poured in 30 ml of cold water and neutralized by 50 % concentration of conc. HCl by volume to volume, a precipitate was collected by filtration and washed with cold water. The isolated obtained products were present in pure form and did not require any further purification. In most of the cases, products were above 98 % pure as judged by 1H NMR.

Spectroscopic data

4-Hydroxy-2-methyl-6-(4-nitrophenyl)pyrimidine-5-carbonitrile (AC1)

Brown solid, Mp: 270–272 °C, yield: 91 %. 1H NMR (300 MHz, DMSO-d 6 ) δ (ppm): 2.46 (s, 3H, –CH3), 8.11 (d, 2H, J = 6 Hz, Ar–H), 8.40 (d, 2H, J = 6 Hz, Ar–H), 13.61 (br.s, 1H, –OH). 13C NMR (75 MHz, DMSO-d 6) δ (ppm): 22.01, 96.84, 115.13, 123.66, 130.06, 141.30, 148.91, 160.46, 163.62, 167. 08. IR (ATR): 3066, 2873, 2231, 1649, 1584, 1525, 1476, 11212, 1166, 1045, 840, 793, 653, 699 cm−1. Anal. Calcd. (%) for C12H8N4O3: C, 56.25; H, 3.15; N, 21.87. Found: C, 56.23; H, 3.17; N, 21.85.

4-Hydroxy-2-methyl-6-(3-nitrophenyl)pyrimidine-5-carbonitrile (AC2)

Yellow solid, Mp: 280–282 °C, yield: 93 %. 1H NMR (300 MHz, DMSO-d 6 ) δ (ppm): 2.47 (s, 3H, –CH3), 7.88 (t, 1H, J = 6 Hz, Ar–H), 8.35 (d, 1H, J = 6 Hz, Ar–H), 8.45 (d, 1H, J = 6 Hz, Ar–H), 8.67 (s, 1H, Ar–H), 13.60 (br.s, 1H, –OH). 13C NMR (75 MHz, DMSO-d 6) δ (ppm): 21.99, 96.44, 115.25, 123.31, 125.98, 130.38, 134.76, 136.79, 147.71, 160.51, 163.61, 166.49. IR (ATR): 2802, 2226, 1677, 1591, 1527, 1423, 1349, 1317, 1112, 1037, 865, 846, 733 cm−1. MS (m/z): 256. Anal. Calcd. (%) for C12H8N4O3: C, 56.25; H, 3.15; N, 21.87. Found: C, 56.28; H, 3.11; N, 21.90.

4-(3,4-Dimethoxyphenyl)-6-hydroxy-2-methylpyrimidine-5-carbonitrile (AC3)

Brown solid, Mp: 278–280 °C, yield: 94 %. 1H NMR (300 MHz, DMSO-d 6 ) δ (ppm): 2.43 (s, 3H, –CH3), 3.81 (s, 3H, –OCH3), 3.85 (s, 3H, –OCH3), 7.14 (d, 1H, J = 6 Hz, Ar–H), 7.56 (s, 1H, Ar–H), 7.62 (d, 1H, J = 9 Hz, Ar–H), 13.30 (br.s 1H, –OH). 13C NMR (75 MHz, DMSO-d 6 ) δ (ppm): 22.46, 56.02, 56.17, 94.10, 111.69, 112.33, 116.71, 123.09, 127.75, 148.69, 152.33, 161.58, 162.75. 168.35. IR (ATR): 2832, 2221, 1655, 1586, 1514, 1264, 1243, 1155, 1015, 783, 770, 672 cm−1; MS (m/z): 271. Anal. Calcd. (%) for C14H13N3O3: C, 61.99; H, 4.83; N, 15.49. Found: C, 61.97; H, 4.86; N, 15.53.

4-(4-Chlorophenyl)-6-hydroxy-2-methylpyrimidine-5-carbonitrile (AC4)

Brown solid, Mp: 180–182 °C, yield: 92 %. 1H NMR (300 MHz, DMSO-d 6 ) δ (ppm): 2.43 (s, 3H, –CH3), 7.53 (d, 2H, J = 9 Hz, Ar–H), 7.91 (d, 2H, J = 9 Hz, Ar–H), 13.49 (br,s 1H, –OH). 13C NMR (75 MHz, DMSO-d6) δ (ppm): 22.32, 96.00, 115.62, 128.90, 130.70, 134.35, 137.26, 161.15, 163.04, 167.95 IR (ATR): 2838, 2227, 1658, 1594, 1584, 1540, 1485, 1259, 1033, 785, 712, 666 cm−1. MS (m/z): 245. Anal. Calcd. (%) for C12H8 ClN3O: C, 58.67; H, 3.28; 14.43; N, 17.10. Found: C, 58.64; H, 3.30; N, 17.13.

4-(4-Bromophenyl)-6-hydroxy-2-methylpyrimidine-5-carbonitrile (AC5)

Brown solid, Mp: 183–185 °C, yield: 95 %. 1H NMR (300 MHz, DMSO-d 6 ) δ (ppm): 2.44 (s, 3H, –CH3), 7.78 (d, 2H, J = 9 Hz, Ar–H), 7.84 (d, 2H, J = 9 Hz, Ar–H), 13.50 (br.s 1H, –OH). 13C NMR (75 MHz, DMSO-d6) δ (ppm): 22.29, 95.97, 115.53, 126.07, 130.76, 131.79, 134.60, 161.21, 163.05, 167.14, 168.05. IR (ATR): 3102, 2220, 1660, 1588, 1526, 1414, 1246, 1085, 1027, 780, 715, 663 cm−1. Anal. Calcd. (%) for C12H8 BrN3O: C, 49.68; H, 2.78; N, 14.48. Found: C, 49.65; H, 2.80; N, 14.51.

4-Hydroxy-2-methyl-6-(3,4,5-trimethoxyphenyl)pyrimidine-5-carbonitrile (AC6)

Brown solid, Mp: 240–242 °C, yield: 92 %. 1H NMR (300 MHz, DMSO-d 6 ) δ (ppm): 2.43 (s, 3H, –CH3), 3.78 (s, 3H, –OCH3), 3.84 (s, 6H, (–OCH3)2), 7.24 (s, 2H, Ar–H), 13.38 (br.s, 1H, –OH). 13C NMR: (75 MHz, DMSO-d 6 ) δ (ppm): 22.28, 56.30, 60.67, 95.25, 106.69, 116.01, 130.47, 140.92, 152.91, 161.48, 162.29, 168.47. IR (ATR): 2826, 2218, 1654, 1583, 1506, 1462, 1362, 1308, 1122, 999, 786, 724. MS (m/z): 301. Anal. Calcd. (%) for C15H15N3O4: C, 59.79; H, 5.02; N, 13.95. Found: C, 59.76; H, 5.06; N, 13.91.

4-Hydroxy-6-(3-methoxyphenyl)-2-methylpyrimidine-5-carbonitrile (AC7)

Yellow solid, Mp: 220–222 °C, yield: 93 %. 1H NMR (300 MHz, DMSO-d 6 ) δ (ppm): 2.43(s, 3H, –CH3), 3.81 (s, 3H, –OCH3), 7.18 (t, 1H, J = 3 Hz, Ar–H), 7.41 (s, 1H, Ar–H), 7.47 (d, 2H, J = 6 Hz, Ar–H), 13.45 (br.s, 1H, –OH). 13C NMR: (75 MHz, DMSO-d 6 ) δ (ppm): 22.24, 55.53, 95.98, 114.06, 115.65, 117.67, 121.22, 129.81, 136.77, 159.43, 161.41, 162.86, 169.11. IR (ATR): 3089, 2228, 1679, 1578, 1426, 1287, 1071, 1010, 833, 788, 728, 653 cm−1. Anal. Calcd. (%) for C13H11N3O2: C, 64.72; H, 4.60; N, 17.42. Found: C, 64.59; H, 4.58; N, 17.44.

4-Hydroxy-6-(4-methoxyphenyl)-2-methylpyrimidine-5-carbonitrile (AC8)

Brown solid, Mp: 234–236 °C, yield: 91, 1H NMR (300 MHz, DMSO-d 6 ) δ (ppm): 2.41 (s, 3H, –CH3), 3.84 (s, 3H, –OCH3), 7.01 (d, 2H, J = 9 Hz, Ar–H), 7.96 (d, 2H, J = 6 Hz, Ar–H), 13.28 (br.s, 1H, –OH). 13C NMR (75 MHz, DMSO-d 6 ) δ (ppm): 22.28, 55.69, 94.13, 114.06, 114.60, 116.27, 127.77, 131.06, 132.00, 161.57, 162.17, 162.52, 168.27. IR (ATR): 3086, 2228, 1682, 1580, 1428, 1387, 1093, 1014, 835, 789, 731, 654 cm−1. MS (m/z): 241. Anal. Calcd. (%) for C13H11N3O2: C, 64.72; H, 4.60; N, 17.42. Found: C, 64.75; H, 4.57; N, 17.45.

4-[4-(Dimethylamino)phenyl]-6-hydroxy-2-methylpyrimidine-5-carbonitrile (AC9)

Greenish solid, Mp: 270–272 °C, yield: 92 %,1H NMR (300 MHz, DMSO- d 6 ) δ (ppm): 2.39 (s, 3H, –CH3), 3.03 (s, 6H, –N(CH3)2), 6.80 (d, 2H, J = 9 Hz, Ar–H), 7.98 (d, 2H, J = 9 Hz, Ar–H), 13.02 (br.s, 1H, –OH). 13C NMR (75 MHz, DMSO-d 6 ) δ (ppm): 22.25, 90.83, 111.22, 117.31, 121.36, 131.00, 153.04, 161.62, 162.19, 167.80. IR (ATR): 3140, 2199, 1610, 1391, 1248, 1150, 1016, 929, 884, 745 cm−1. Anal. Calcd. (%) for C14H14N4O: C, 66.13; H, 5.55; N, 22.03. Found: C, 66.09; H, 5.52; N, 22.00.

4-Hydroxy-2-methyl-6-(4-methylphenyl)pyrimidine-5-carbonitrile (AC10)

Yellow solid, Mp: 232–234 °C, yield: 93 %. 1H NMR (300 MHz, DMSO-d 6 ) δ (ppm): 2.37 (s, 3H, –CH3), 2.42 (s, 3H, –CH3), 7.25 (d, 2H, J = 9 Hz, Ar–H), 7.81 (d, 2H, J = 6 Hz, Ar–H), 13.18 (br.s, 1H, –OH). 13C NMR (75 MHz, DMSO- d 6 ) δ (ppm): 21.49, 22.26, 95.08, 116.10, 132.70, 142.43, 161.53, 163.00, 169.21. IR (ATR): 2837, 2220, 1658, 1584, 1510, 1377, 1250, 1181, 1023, 845, 790, 669 cm−1. Anal. Calcd. (%) for C13H11N3O: C, 69.32; H, 4.92; N, 18.66. Found: C, 69.28; H, 4.94; N, 18.64.

Biology

Anti-inflammatory activity

The anti-inflammatory activity of the synthesized pyrimidine derivatives have been determined by carrageenan-induced rat paw edema assay.

Animals

Wistar rats (150–200 g) of either sex were group housed (n = 6) under a standard 12-h light–dark cycle and under controlled conditions of temperature and humidity (25 ± 2 °C, 55–65 %). The rats were given standard rodent chow and water ad libitum. They were acclimatized to laboratory conditions for 7 days before carrying out the experiments. All the experiments were carried out in a noise-free room between 08.00 and 15.00 h. A separate group (n = 6) of rats was used for each set of experiments.

Toxicity study

The preliminary experiments were carried out on rats (n = 6). The synthesized compounds AC1 to AC10 were administered orally in different doses to find out the range of doses which cause 0 and 100 % mortality of animals. A range doses were determined for each compound. In ten groups, compounds AC1 to AC10 were given p.o. in doses of 100, 200, 250, 300, 400, and 500 mg/100 g b.w. The LD50 was evaluated by Spearman and Karber method [47]. The test compounds were administrated p.o. at different doses and the animals were kept under observation for mortality as well as any behavioral changes for evaluation of a possible anti-inflammatory activity.

Carrageenan induced rat paw edema assay

The animals were divided into five groups of six animals each and were fasted for a period of 24 h prior to the study. The group first was treated as control (0.1 ml of 1 % (w/v) of carrageenan subcutaneously), group second was given diclofenac sodium 30 mg/kg, p.o. and the remaining groups were treated with 25 and 50 mg/kg, p.o. of compounds AC1-10. The edema was induced by injecting 0.1 ml of a 1 % solution of carrageenan in saline into the sub-plantar region of the right hind paw of the rats. The volumes of edema of the injected and the contralateral paws were measured at 1-h intervals for up to 5 h using a plethysmograph. The percentage inhibition was calculated by using following equation.

$${\text{Percentage}}\;{\text{inhibition}} = \frac{{V_{\text{c}} - V_{\text{t}} }}{{V_{\text{c}} }} \times 100$$

where V c—edema volume of control group, V t—edema volume of test group.

Statistical analysis

All analysis was performed using GraphPad Prism for Windows (GraphPad Software Inc., La Jolla, CA, USA). All statistical analysis is expressed as mean ± standard error of the mean (SEM). The data were analyzed by one-way analysis of variance (ANOVA), where applicable p < 0.05 was considered statistically significant, compared with vehicle followed by Dunnett’s test.